Chronic liver disease is now a leading cause of death and adult disease in Korea. Liver has a big buffering capacity but once liver disease is developed it does not recognized until it is significantly progressed because there are almost no self-detected symptoms in the early stage of the disease. Make matter worse, no effective therapeutic agent or diagnostic method has been established. Causes of liver disease are alcohol, drugs, chemicals, viral hepatitis, metabolic disease such as biliary track disease and auto-immune disease but there are still more causes unknown. In the meantime, the liver seems to overwork these days as a center of detoxication, blood storage and circulation and the regulation of blood volume, because modern people are more exposed on harmful industrial materials, asking more detoxication, and excessive stresses, alcohol over-drinking, smoking, causing liver damage and further resulting in malfunctioning in immune system and being a cause for another disease.
Complications and hepatoma affecting prognosis in most chronic liver diseases are detected when liver injury is progressed into hepatic fibrosis and hepatic cirrhosis. Therefore, it is important to understand the progress of hepatic fibrosis and to develop a therapeutic agent inhibiting the progress. The therapeutic agents for liver disease in clinical trail are Silymarin produced by Madaus Co., Germany, in 1970s, Ara-AMP produced by Park Davis Co., USA, Carbaica provided by Selvi Co., Italy and DDB provided by Institute of Chinese Materia Medica, China in 1980s, but these drugs carry serious side effects and exhibit low therapeutic effect. Thus, more economical, safe and effective therapeutic agent is in urgent need.
Hepatic fibrosis and liver cirrhosis have been known to be caused by interaction between cytokine and extracellular matrix (ECM). Damaged liver cells induce phagocytosis by kupffer cells, and thus activated kupffer cells secret various cytokines to activate hepatic stellate cells, which has been regarded as a general mechanism in the liver but recent reports add that the damaged liver cells directly activate hepatic stellate cells, so called autocrine. That is, once apoptosis occurs by liver cell damage caused at any reason, TGF-1 is expressed to activate hepatic stellate cells, and then the activated hepatic stellate cells induce TGF-1 expression again that causes apoptosis and this vicious cycle is presumably a cause for serious liver disease (Sun F, et al., Biochim Biophys Acta. 2001 Feb. 14; 1535(2):186-191; Jeong W I, et al., Liver Int. 2004 December; 24(6):248-254; Marcin Stopa, et al., J. Biol. Chem. 2000 September; 29308-29317; Jeong W. I, et al., Anticancer Res. 2002 March-April; 22(2A):869-877; Ishak kg, et al., Alcohol Clin Exp Res. 1991; 15:45-66; Korean Patent No. 2001-0036463).
To screen a pharmaceutical composition for the prevention of liver dysfunction, studies have been undergoing using microorganisms, plants, and synthetic chemicals, but it still has a long way to go for industrialization, leaving problems including difficulty in obtaining target materials.
The present inventors tried to find a novel protease and as a result the inventors separated a novel microorganism Aranicola proteolyticus HY-3 strain (Accession No: KCTC 0268BP; WO 01/57222) from Nephila clavata. The present inventors then separated a novel protease Arazyme from the said novel strain. Arazyme not only exhibits excellent protein degradation activity at low temperature and at high salt concentration but also is highly activated at human body temperature of 37° C., in addition to exhibiting very stable activity in wide pH range. The present inventors further identified the gene of this novel protease (WO 01/57222).
The present inventors investigated the effects of arazyme originated from Aranicola proteolyticus and completed this invention by confirming that arazyme can protect liver from damages and thereby can be effectively used as a pharmaceutical composition for the prevention of liver dysfunction.